Strategies for accurate quantitation of free payloads in antibody-drug-conjugates: application for a payload with a labile lactone group.

Quantifying free payloads in biological matrices is essential for understanding Antibody-Drug Conjugates (ADC) off-target toxicity and safety. The bioanalysis of payloads is challenging due to the need to measure trace amount amidst abundant ADC, with minor ADC degradation potentially causing substantial payload overestimation. Successful assays require careful evaluation of payload structures and effective management of ADC-related interferences. This study identifies challenges for lactone-containing free payloads, mitigates their impact on bioanalysis, and develops a validated methodology for accurate measurement of these payloads in human serum. Lactone hydrolysis and its formation from carboxylate were evaluated at various pH values in buffer solutions and serum. The lactone was completely hydrolyzed at pH 8.5 (25°C) within 25 min; it took several hours at pH 7 and was stable at pH ≤ 6. Lactone regeneration from carboxylate was rapid at pH 3 (within 5 min) and slower at pH ≥ 4. In human serum, lactone hydrolysis was relatively fast (approximately 2 h at 37°C), suggesting the carboxylate form predominates in circulation. Stability experiments showed lactone hydrolysis in serum is reversible, eliminating the need for sample treatment at clinical sites. These insights were applied in the design of a method based on protein precipitation and solid-phase extraction to quantify total payload exposure (50-10,000 pg/mL) in serum in the presence of ADC (250 µg/mL). A double liquid-liquid extraction was employed to purify the ADC before use to prevent interferences in the selectivity and stability assessment. The assay was validated according to M10 guidance and used to support clinical studies.